By gene analysis using Drosophila, it became clear that Oskar, Vasa, Tudor, and Nanos genes have core functions in the determination mechanism of germ cells (for example, Rongo, C., et al., Development, 121, 2737-2746, 1995). These genes are all accumulated in polar granule in the formation of egg cells, and this blastomere having a maternal determination factor determines germ cell destiny. It is considered that Vasa gene encodes ATP-dependent RNA helicase and that its functions are associated with regulation of translation from mRNA to a protein (for example, Liang, L., et al, Development, 120, 1201-1211, 1994). In addition, a structure for its enzymatic function is evolutionally strongly conserved. Thus, Vasa homolog genes have been identified in many multicellular animal species ranging from Platyhelminthes (planarian) to humans.
Based on the aforementioned findings, as a method for simply sorting a cell having germ cell differentiation potency using, as an indicator, the expression of a marker gene, without performing complicated operations such as homologous recombination, there has been reported a method for obtaining a germ cell, which comprises recovering a cell having germ cell differentiation potency from a transgenic non-human mammal, into which a recombinant expression vector comprising a marker gene incorporated therein such that it is under the control of the promoter sequence of a Vasa homolog gene derived from the mammal has been introduced, using the expression of the marker gene as an indicator (for example, Japanese Laid-Open Patent Application Nos. 2006-333762 and 2003-235558).
On the other hand, primordial germ cell is an original cell for egg and sperm, which is modified to an individual via processes of maturation and fertilization. There has been known a method for inducing the differentiation of a separated primordial germ cell derived from fish into a germ cell line, which comprises transplanting the fish-derived separated primordial germ cell into the early embryo of a recipient fish of a different species, and particularly transplanting the separated primordial germ cell into the peritoneal cavity of a recipient fish of a different species at the early development stage (for example, Japanese Patent Laid-Open Nos. 2006-333762 and 2003-235558).
At present, in cultivation of tuna, a method in which native juvenile fish (in general, several tens of to several hundreds of gram) are captured by a fisher and grown has been mainly applied. In recent years, the amount of native tuna caught has been reduced, and thus catch quotas for mature tuna have been strictly limited. Hence, a stable supply of juvenile fish would not be guaranteed in the future by a method of obtaining such juvenile fish from nature. In addition, as in the case of salmon and Pagrus major, if a technique of artificial seedling production was established, it would be expected that breeding can be carried out by alternation of generations, while selecting parent fish having good traits, and that juvenile fish having stable quality can be supplied with better cultivation efficiency. The mechanism of maturation of tuna has not yet been sufficiently clarified. However, it is considered that tuna reaches initial maturation after its body weight has exceeded several tens of kilograms. Since the body size of tuna is large, differing from other fish species, it is grown by a seedling production by a method of collecting fertilized eggs naturally laid by parent fish in a preserve or in an enclosed bay using a finely-woven net. Since Pagrus major and the like lay eggs in a water tank, a device for collecting the eggs with a net by overflowing seawater on the surface of the tank can be easily produced. However, when such operation is carried out at sea, it is very troublesome.
When specific individual fishes are to be mated for the purpose of breeding or the like, artificial egg collection is carried out by squeezing the abdomen of a mother fish, sperm is also collected in the same manner, and the collected egg and sperm are then subjected to artificial insemination. However, in a case in which parent fishes are large in size, like tuna, this method is not easy. Moreover, in the industrial field, for the purpose of controlling shipment time and fish cultivation period, it is possible to enhance profitability by shifting the time at which juvenile fish is produced. Therefor, it is necessary to control water temperature and photoperiod in a place where parent fishes can be environmentally controlled, so as to shift the season, thereby controlling the time at which the parent fishes lay eggs. However, in a case in which parent fishes are large in size, like tuna, enormous manpower and costs are required.
Surrogate fish technique is a technique of allowing fish species that are suitable for seedling production to produce the gametes of fish species that are unsuitable for seedling production, or to lay eggs and then to be subjected to insemination, so as to simply allow seedling production at low costs. For example, if the surrogate fish technique described in the aforementioned Patent Document 2 is applied to tuna, so as to allow small-sized fish species used as recipient fishes to maturate tuna-derived germ cells, full cultivation including seedling production can be achieved in a small water tank, and it is expected to result in significant laborsaving and cost reduction. In transplanting a separated primordial germ cell, it is necessary to propagate tuna-derived primordial germ cells incorporated into the gonad of a recipient and to detect the ratio between recipient-derived germ cells and donor-derived germ cells. It is an object of the present invention to provide a method for inducing the differentiation of a primordial germ cell into a germ cell line, which comprises transplanting a primordial germ cell derived from a Perciformes donor fish such as a tuna into the early embryo of a recipient fish of a different species, wherein ovum and/or sperm derived from the donor fish are specifically detected, and such donor fish-derived ovum and/or sperm are then distinguished from germ cells derived from the recipient fish.
The present inventors have succeeded in producing a rainbow trout from a masu salmon (Oncorhynchus masou) by carrying out heteroplastic germ cell transplantation on Salmonidae fish. In this transplantation, a genetically modified fish line in which the germ cell of a rainbow trout had been visualized with a green fluorescent protein was used, and as a result, it became possible to easily confirm the success or failure of the transplantation. In addition, in order to apply such heteroplastic germ cell transplantation to native, endangered fish species or cultured fish species, a method for confirming the success or failure of the transplantation without using a genetically modified fish line has already been developed. By this method, the present inventors have succeeded in detecting wild-type rainbow trout germ cells surviving at the genital gland of a Salvelinus pulvius host. The present inventors aim to apply this heteroplastic germ cell transplantation method to other marine fish species. However, to realize this object, it is essential to develop a method for confirming whether or not the transplanted germ cells of a Perciformes donor fish such as a tuna have been incorporated into the genital gland of a host and they survive therein.
In order to develop such method, the present inventors have selected Vasa gene from among Nanos gene, Deadend gene, Vasa gene, and other genes, which had been known to be specifically expressed in primordial germ cells. Thereafter, the inventors have determined for the first time the nucleotide sequences of the Vasa genes of a tuna, a chub mackerel, a spotted mackerel, an eastern little tune, and a drum fish. Further, the inventors have focused on a tuna Vasa gene, which is most likely to become a Perciformes donor fish, and they have confirmed that such tuna Vasa gene is specifically expressed in the primordial germ cell and spermatogonium/oogonium of a tuna. At the same time, in order to avoid incorrect detection of a drumfish Vasa gene having extremely high homology with the tuna Vasa gene, the inventors have specified a region characteristic for the tuna Vasa gene, and thus they have found that this region can be used as an identification marker for a spermatogonium/an oogonium derived from tuna primordial germ cells. Moreover, in order to analyze tuna germ cells transplanted into the genital gland of a host, it is essential to establish a method of distinguishing a tuna Vasa gene from a host Vasa gene and then detecting only the tuna gene. However, since the nucleotide sequences of the Vasa genes of fish species are extremely highly homologous with one another, it had been difficult to design a PCR primer set for specifically detecting the expression of a tuna Vasa gene. Thus, the inventors of the present application have carried out nested PCR that enables highly specific amplification from a trace amount of DNA, so that they could specifically detect a tuna Vasa gene. Furthermore, the inventors have compared the sequence of a tuna Vasa gene with the sequence of a Vasa gene of another Perciformes fish, and as a result, they have specified a restriction enzyme sequence existing only in the tuna Vasa gene. By combining such nested PCR with a restriction enzyme treatment, the present inventors have established a method for more reliably detecting a tuna Vasa gene, thereby completing the present invention.
Specifically, the present invention relates to    (1) a protein consisting of the amino acid sequence shown in SEQ ID NO 2 of the sequence listing; a protein, which consists of an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or several amino acids with respect to the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, and which is specifically expressed in a tuna germ cell; or a protein, which consists of an amino acid sequence having homology of at least 85% or more with the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, and which is specifically expressed in a tuna germ cell,    (2) a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing; a protein, which consists of an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or several amino acids with respect to the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, and which is specifically expressed in a tuna germ cell; or a protein, which consists of an amino acid sequence having homology of at least 85% or more with the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, and which is specifically expressed in a tuna germ cell, and    (3) a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 of the sequence listing; a DNA, which hybridizes under stringent conditions with a DNA consisting of a sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 of the sequence listing, and which encodes a protein specifically expressed in a tuna germ cell; a DNA, which hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence having a function as a primer or a probe produced from a portion of the nucleotide sequence shown in SEQ ID NO: 1 of the sequence listing, and which encodes a protein specifically expressed in a tuna germ cell; or a DNA, which consists of a nucleotide sequence comprising a substitution, deletion, insertion, or addition of one or several nucleotides with respect to the nucleotide sequence shown in SEQ ID NO: 1 of the sequence listing, and which encodes a protein specifically expressed in a tuna germ cell.
In addition, the present invention relates to    (4) a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 8, or 10 of the sequence listing; a protein, which consists of an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or several amino acids with respect to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, or 10 of the sequence listing, and which is specifically expressed in the germ cell of a Perciformes fish; or a protein, which consists of an amino acid sequence having homology of at least 85% or more with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, or 10 of the sequence listing, and which is specifically expressed in the germ cell of a Perciformes fish,    (5) a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, 6, 8, or 10 of the sequence listing; a protein, which consists of an amino acid sequence comprising a substitution, deletion, insertion, or addition of one or several amino acids with respect to the amino acid sequence shown in SEQ ID NO: 4, 6, 8, or 10 of the sequence listing, and which is specifically expressed in the germ cell of a Perciformes fish; or a protein, which consists of an amino acid sequence having homology of at least 85% or more with the amino acid sequence shown in SEQ ID NO: 4, 6, 8, or 10 of the sequence listing, and which is specifically expressed in the germ cell of a Perciformes fish, and    (6) a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3, 5, 7, or 9 of the sequence listing; a DNA, which hybridizes under stringent conditions with a DNA consisting of a sequence complementary to the nucleotide sequence shown in SEQ ID NO: 3, 5, 7, or 9 of the sequence listing, and which encodes a protein specifically expressed in the germ cell of a Perciformes fish; a DNA, which hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence having a function as a primer or a probe-produced from a portion of the nucleotide sequence shown in SEQ ID NO: 3, 5, 7, or 9 of the sequence listing, and which encodes a protein specifically expressed in the germ cell of a Perciformes fish; or a DNA, which consists of a nucleotide sequence comprising a substitution, deletion, insertion, or addition of one or several nucleotides with respect to the nucleotide sequence shown in SEQ ID NO: 3, 5, 7, or 9 of the sequence listing, and which encodes a protein specifically expressed in the germ cell of a Perciformes fish.
Moreover, the present invention relates to    (7) a recombinant vector comprising the DNA according to (2), (3), (5), or (6),    (8) a transformant transformed with the recombinant vector according to (7),    (9) a fusion protein or fusion peptide, or salt thereof obtained by binding the protein according to (1) or (4) with a marker protein and/or a peptide tag,    (10) an antibody against the protein according to (1) or (4), or the fusion protein or fusion peptide according to (9), or salt thereof, and    (11) a primer set or a probe for detecting the presence of a DNA and/or mRNA encoding the protein according to (1) or (4).
Furthermore, the present invention relates to    (12) a method for detecting a primordial germ cell, a spermatogonium, or an oogonium derived from a Perciformes donor fish, which has been transplanted into a recipient fish of a different species, which methods comprises using the primer set or probe according to (11),    (13) the detection method according to (12), which comprises: treating a DNA fragment amplified by PCR using the primer set according to (11) with at least one restriction enzyme; and determining whether or not the amplified DNA fragment is derived from the Perciformes donor fish, using the length of the digested or undigested DNA fragment as an indicator,    (14) the detection method according to (12) or (13), wherein the Perciformes donor fish is a tuna,    (15) the detection method according to (14), wherein the primer set is designed to amplify a region comprising a restriction enzyme HpaI recognition sequence existing in a DNA encoding the protein according to (1); and which method comprises treating a DNA fragment amplified by PCR using the primer set with HpaI, and determining that the DNA fragment is derived from bluefin tuna DNA, when it is digested, and    (16) the detection method according to (15), wherein the PCR is nested PCR using a first primer set consisting of the nucleotide sequences shown in SEQ ID NOS: 19 and 20 and a nested primer set consisting of the nucleotide sequences shown in SEQ ID NOS: 21 and 22.
Still further, the present invention relates to    (17) a method for detecting a primordial germ cell, a spermatogonium, or an oogonium derived from a Perciformes donor fish, which has been transplanted into a recipient fish of a different species, which method comprises using the antibody according to (10),    (18) the detection method according to (17), wherein the Perciformes donor fish is a tuna,    (19) a method for evaluating the growth and/or maturation of a tuna germ cell derived from a Perciformes donor fish transplanted into a recipient fish of a different species, which comprises the detection method according to any one of (12) to (18), and    (20) the evaluation method according to (19), wherein the Perciformes donor fish is a tuna.